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West Nile virus in Tunisia, 2014: First isolation from mosquitoes
Acta Tropica 159 (2016) 106–110
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short communication
West Nile virus in Tunisia, 2014: First isolation from mosquitoes F. Wasfi a , K. Dachraoui a , S. Cherni a , A. Bosworth b , W. Barhoumi a , S. Dowall b , I. Chelbi a , M. Derbali a , Z. Zoghlami a , J.C. Beier c , E. Zhioua a,∗ a b c
Institut Pasteur de Tunis, Laboratory of Vector Ecology, 13 Place Pasteur, 1002 Tunis, Tunisia Public Health England, Porton Down, Salisbury, Wiltshire SP4 0JG, UK Department of Public Health Sciences, University of Miami Miller School of Medicine, Miami, FL 33136, USA
a r t i c l e
i n f o
Article history: Received 11 December 2015 Received in revised form 29 March 2016 Accepted 30 March 2016 Available online 31 March 2016 Keywords: Flaviviruses West Nile virus Culex mosquitoes Emerging North Africa
a b s t r a c t Several outbreaks of human West Nile virus (WNV) infections were reported in Tunisia during the last two decades. Serological studies on humans as well as on equine showed intensive circulation of WNV in Tunisia. However, no virus screening of mosquitoes for WNV has been performed in Tunisia. In the present study, we collected mosquito samples from Central Tunisia to be examined for the presence of flaviviruses. A total of 102 Culex pipiens mosquitoes were collected in September 2014 from Central Tunisia. Mosquitoes were pooled according to the collection site, date and sex with a maximum of 5 specimens per pool and tested for the presence of flaviviruses by conventional reverse transcription heminested PCR and by a specific West Nile virus real time reverse transcription PCR. Of a total of 21 pools tested, 7 were positive for WNV and no other flavivirus could be evidenced in mosquito pools. In addition, WNV was isolated on Vero cells. Phylogenetic analysis showed that recent Tunisian WNV strains belong to lineage 1 WNV and are closely related to the Tunisian strain 1997 (PAH 001). This is the first detection and isolation of WNV from mosquitoes in Tunisia. Some areas of Tunisia are at high risk for human WNV infections. WNV is likely to cause future sporadic and foreseeable outbreaks. Therefore, it is of major epidemiological importance to set up an entomological surveillance as an early alert system. Timely detection of WNV should prompt vector control to prevent future outbreaks. In addition, education of people to protect themselves from mosquito bites is of major epidemiological importance as preventive measure against WNV infection. © 2016 Published by Elsevier B.V.
1. Introduction In the Western Mediterranean basin, West Nile virus (WNV) is transmitted mainly by Culex mosquitoes (Turell, 2012; Vázquez et al., 2011). In natural foci, the circulation of WNV involves birds as amplifying host and bird-feeding mosquitoes (Jourdain et al., 2007) with humans and equines as incidental hosts. WNV is widely distributed in Africa, Middle East, Asia, Southern Europe and the Americas (Hubálek and Halouzka, 1999). A specific WNV lineage 1 variant was also reported in Australia (Scherret et al., 2001). Most of human WNV infections are asymptomatic with less than 1% of infected individuals developing severe neuroinvasive diseases such as meningitis, encephalitis and flaccid paralysis (Kramer et al., 2007).
In Tunisia, several major outbreaks of human WNV infections occurred during 1997 (Triki et al., 2001), 2003 (Hachfi et al., 2010; Riabi et al., 2014), and 2012 (Bouatef et al., 2012). Sporadic cases were reported in 2007, 2010, and 2011 (Bouatef et al., 2012). Several studies showed an intensive circulation of WNV among humans (Riabi et al., 2010; Bahri et al., 2011), equines (Ben Hassine et al., 2014) and birds in Tunisia (Hammouda et al., 2015). A risk map for WNV infection in equines in Tunisia showed that the governorates of Jendouba, Nabeul, Sousse, Monastir, Sfax, Mednine and Djerba are considered as high-risk areas (Bargaoui et al., 2015). To date, no virological data concerning WNV in mosquitoes for Tunisia are available. In the present study, we performed a preliminary virological screening of mosquitoes for WNV in Tunisia. 2. Materials and methods
∗ Corresponding author at: Institut Pasteur de Tunis, Laboratory of Vector Ecology, 13 Place Pasteur BP 74, 1002 Tunis, Tunisia. E-mail addresses: [email protected], [email protected] (E. Zhioua). http://dx.doi.org/10.1016/j.actatropica.2016.03.037 0001-706X/© 2016 Published by Elsevier B.V.
2.1. Study sites and mosquito samplings The study was performed in two villages [(Saddaguia: 35◦ 05 N, 9◦ 25 E and El Felta: 35◦ 16 N, 9◦ 26 E)] belonging to the governorate
F. Wasfi et al. / Acta Tropica 159 (2016) 106–110
107
Fig. 1. Bioclimatic map showing mosquitoes capture sites in Tunisia.
of Sidi Bouzid located in Central Tunisia with an arid bio-climate (Fig. 1). During the last 20 years, environmental changes due to irrigation have occurred in these arid areas located in Cen-
tral Tunisia and lead to the establishment of sand fly vectors of zoonotic visceral leishmaniasis previously limited to the humid areas located in Northern Tunisia (Barhoumi et al., 2015), and to
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F. Wasfi et al. / Acta Tropica 159 (2016) 106–110
intense circulation of sandfly-borne phleboviruses (Fares et al., 2015). Human-induced habitat change such as the development of irrigation in arid and semi-arid areas of North Africa was shown to have a direct impact on the geographical distribution of C. pipiens (Conley et al., 2014). Within this context, we also investigated the presence of flaviviruses in mosquito populations in these highly environmentally-disturbed areas. Mosquito sampling was performed by using CDC light-traps during September 2014 (September 9, 10, and 23). CDC light-traps were placed inside houses and in animal shelters located in the peri-domestic areas to collect endophilic and exophilic mosquito species. Live mosquitoes were morphologically identified to species level using the identification keys (Brunhes et al., 2000) and were pooled according to the collection site, date, species and sex with a maximum of 5 specimens per pool and directly stored at in −80 ◦ C until use. 2.2. Viral detection, sequencing and phylogenetic analysis Mosquito pools were manually ground in 500 l of Minimum Essentiel Medium (MEM) supplemented with antibiotic solution. The mixture was clarified by centrifugation at 6000g for 2 min. Viral RNA was extracted from 140 l of mosquito homogenates using the QIAmp Viral RNA Mini Kit (Qiagen, Hilden, Germany). During the RNA extraction, no positive control was used to preclude any risk of contamination. Instead, only negative control consisting of water solution was used. RNA extracts were tested by conventional reverse transcription heminested PCR (Scaramozzino et al., 2001) and by a specific West Nile virus real time reverse transcription PCR (Linke et al., 2007). West Nile virus RNA of the Tunisian strain (PaH001) isolated from a deceased patient in 1997 was used as positive control (Triki et al., 2001; Charrel et al., 2003). PCR amplification products were sequenced directly using the Big Dye Terminators v3.1 kit (Applied Biosystems). Sequences of heminested PCR product were firstly aligned together with 23 sequences of selected flaviviruses and with 38 sequences of West Nile virus retrieved from the Genbank database using CLUSTAL W 1.4 implemented in MEGA v.5.22 (Tamura et al., 2007). Phylogenetic analysis was performed based on 213-nt of the NS5 viral polymerase gene. The phylogenetic tree was obtained by using the maximum likelihood analysis method and the tamura-3 model with general time-reversible rates among sites. The tree topology was supported by 500 bootstrap replicates. The Usutu (UsuV) sequence was used as an out-group with West Nile virus sequences. 2.3. Virus isolation Mosquito homogenates of WNV-PCR positive samples were inoculated into Vero cell culture (Vero: WHO cat N◦ 88020401). Only negative control was used to avoid cytotoxicity and contamination risk. Two passages were performed with the Vero cell line. Cells were monitored daily for 7 days for cytopathic effect (CPE). Heminested PCR was performed after only one passage on cells to confirm the presence of detected flavivirus. 3. Results A total of 102 female mosquitoes were collected during 3 trapping nights. All collected mosquitoes from inside houses and animal shelters located in the peri-domestic areas were identified as Culex pipiens. Of a total of 21 pools, 7 were positive by reverse transcription heminested PCR and by the specific West Nile virus real time reverse transcription PCR yielding a minimum infection rate (MIR) of 6.8% (7/102). All positive mosquitoes were collected from animal shelters located in the peri-domestic areas. All PCR products were sequenced and were identified as WNV by Blast analysis. Only RNA
of lineage 1 was detected. Phylogenetic analysis (Fig. 2) showed that the seven sequences of this study formed a monophyletic group supported by an 81% bootstrap value. These sequences were most closely related to the Italian mosquito WNV strain isolated from mosquitoes in 2008, but clearly distinct from the Tunisian WNV strain PHA001 isolated in 1997. Among seven RT-PCR positive pools, only one mosquito homogenate with the lowest ct value inoculated to Vero cells showed CPE. Subsequently, a second RTPCR performed on cell culture supernatant confirmed the presence of WNV viral RNA.
4. Discussion It appears that C. pipiens from North Africa is exo-endophilic species which is a different behaviour from those of Southern Europe where the mosquitoes is mainly exophilic. More studies concerning the behaviour of C. pipiens from North Africa are needed. This report showed the circulation and first detection of WNV lineage 1 in mosquitoes and the first isolation of WNV lineage 1 from mosquitoes in Tunisia. Our results showed that the strain of WNV clusters with the Italian isolate. It is important to point out that our phylogenetic analysis was performed based on a partial highly conserved region of the NS5 polymerase gene (Scaramozzino et al., 2001) allowing only rapid identification of WNV lineages. However, this method has little power to characterize closely related sequences such as the Israeli and North American WNV strains (Sotelo et al., 2011). The clustering of the Tunisian WNV strains with the Italian isolates strongly suggest that WNV is flowing between Tunisia and Europe particularly Italy along migratory bird routes (Jourdain et al., 2007). Our results suggest that C. pipiens is the main vector of WNV in Tunisia. The MIR reported in Czech Republic, Spain, Portugal, and Italy, were 0.012% (Rudolf et al., 2014), 0.1% (Vázquez et al., 2011), 0.28% (Esteves et al., 2004), and 0.02% (Tamba et al., 2011), respectively. In our study, we reported an unexpectedly high MIR but sample size limitation precluded statistical analysis of these data. It is important to point out that this high MIR was obtained by using two different protocols, and therefore, laboratory contamination is excluded. In order to determine a statistically accepted MIR, several thousands of mosquitoes need to be collected and tested for the presence of flaviviruses. Despite the unexpectedly high MIR found in our study, no human cases were reported during 2014 suggesting an intense circulation of WNV among birds. Our results strongly suggest that WNV remains silent and spillover to humans occurs only under favorable ecological conditions. Thus, understanding of the ecological factors to virus circulation and environmental conditions leading WNV outbreaks is of major importance. Persistence of WNV in overwintering C. pipiens is an important mechanism in the maintenance of this arbovirus (Nasci et al., 2001). Thus, WNV is likely to cause future sporadic and foreseeable outbreaks not only in Tunisia but along the migratory flyways of birds between Africa and Eurasia. In addition, outbreaks of WNV infections in humans were reported in Northern Italy, Southern France, Southern Portugal and Central Europe during the transmission season of 2015 (ECDC, 2015). Our results strongly suggest that irrigation in arid areas of North Africa may lead to the emergence of WNV. This hypothesis is corroborated by the intense WNV activity following the development of irrigation networks to flood rice fields located in Northern Morocco (Figuerola et al., 2009; Schuffenecker et al., 2005). Thus, setting up an entomological surveillance as an early alert system around the Mediterranean Basin to prevent future outbreaks is highly needed. New vector control methods, such as attractive toxic sugar bait, show promise for reducing mosquito populations (Khallaayoune et al., 2013), and subsequently should be evaluated in light of our findings.
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Fig. 2. Phylogenetic analysis including 38 different WNV partial NS5 genes and the 7 WNV identified sequences from Tunisian mosquito pools. The Usutu (UsuV) sequence was used as an out-group. Phylogenetic analysis was performed using the maximum likelihood analysis method and the tamura-3 model with general time-reversible rates among sites. The tree topology was supported by 500 bootstrap replicates.
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Competing interests The authors declare that they have no competing interests. Acknowledgements This study was supported by the Pasteur Institute of Tunis, Tunisia and by Public Health England, Porton, UK. References Bahri, O., Dhifallah, I., Ben Alaya-Bouafif, N., Hekih, H., Gargouri, J., Triki, H., 2011. Sero-epidemiological study of West Nile virus circulation in humans in Tunisia. Bull. Soc. Pathol. Exot. 104, 272–276. Bargaoui, R., Lecollinet, S., Lancelot, R., 2015. Mapping the serological prevalence rate of West Nile fever in equids, Tunisia. Transbound Emerg. Dis. 62, 55–66. Barhoumi, W., Qualls, W.A., Archer, R.S., Fuller, D.O., Chelbi, I., Chern i, S., Derbali, M., Arheart, K.L., Zhioua, E., Beier, J.C., 2015. Irrigation in the arid regions of Tunisia impacts the abundance and apparent density of sandfly vectors of Leishmania infantum. Acta Trop. 141, 73–78. Ben Hassine, T., De Massis, F., Calistri, P., Savini, G., Bel Hadj Mohamed, B., Ranen, A., Di Gennaro, A., Sghaier, S., Hammami, S., 2014. First detection of co-circulation of West Nile and Usutu viruses in equids in the south-west of Tunisia. Transbound Emerg. Dis. 61, 385–389. Bouatef, S., Hogga, N., Ben Dhifallah, I., Triki, H., Ben Alya Bouafif, N., Achour, N., 2012. Monitoring and current situation of meningitis and meningo-encephalitis to West Nile virus in Tunisia. Tun. Rev. Infect. 6, 181–182. Brunhes, J., Rhaim, A., Geoffroy, B., Angel, G., Hervy, J.-P., 2000. Les Culicidae d’Afrique Méditerranéene. Institute for Research Development, Montpellier, France. Charrel, R.N., Brault, A.C., Gallian, P., Lemasson, J.J., Murri, S., Pastorino, B., Zeller, H., de Chesse, R., de Micco, P., de Lamballerie, X., 2003. Evolutionary relationship between Old World West Nile virus strains. Evidence for viral gene flow between Africa the Middle East and Europe. Virol. 315, 381–388. Conley, A.K., Fuller, D.O., Haddad, N., Hassan, A.N., Gad, A.M., Beier, J.C., 2014. Modeling the distribution of the West Nile and Rift Valley fever vector Culex pipiens in arid and semi-arid regions of the Middle East and North Africa. Parasites Vectors 7, 289. European centers for diseases Control and prevention, 2015. Distribution of West Nile fever cases by affected areas, European region and Mediterranean Basin Transmission season 2015. http://ecdc.europa.eu/en/healthtopics/west nile fever/West-Nile-fever-maps/PublishingImages/ECDC WNF Affected current season.png. Esteves, A., Almeida, A.P.G., Galao, R.P., Parreira, R., Piedae, J., Rodrigues, J.C., Sousa, C.A., Novo, M.T., 2004. West Nile virus in Southern Portugal, 2004. Vector-Borne Zoonotic Dis. 5, 410–413. Fares, W., Charrel, R.N., Dachraoui, K., Bichaud, L., Barhoumi, W., Derbali, M., Cherni, S., Chelbi, I., de Lamballerie, X., Zhioua, E., 2015. Infection of sand flies collected from different bio-geographical areas of Tunisia with phleboviruses. Acta Trop. 141, 1–6. Figuerola, J., Baouab, R.E., Soriguer, R., Fassi-Fihri, O., Llorente, F., Jímenez-Clavero, M.A., 2009. West Nile virus antibodies in wild birds Morocco, 2008. Emerg. Infect. Dis. 15, 1651–1653. Hachfi, W., Bougmiza, I., Bellazreg, F., Bahri, O., Kaabia, N., Bahri, F., Letaief, A., 2010. Second epidemic of West Nile virus meningoencephalitis in Tunisia. Med. Mal. Infect. 40, 456–461.
Hammouda, A., Lecollinet, S., Hamza, F., Nasri, I., Neb, A., Selmi, S., 2015. Exposure of resident sparrows to West Nile virus evidenced in South Tunisia. Epidemiol. Infect. 143, 3546–3549. Hubálek, Z., Halouzka, J., 1999. West Nile fever—a reemerging mosquito-borne viral disease in Europe. Emerg. Infect. Dis. 5, 643–650. Jourdain, E., Toussaint, Y., Leblond, A., Bicout, D.J., Sabatier, P., Gauthier-Clerc, M., 2007. Bird species potentially involved in introduction, amplification, and spread of West Nile virus in a Mediterranean wetland, the Camargue (Southern France). Vector-Borne Zoonotic Dis. 7, 15–33. Khallaayoune, K., Qualls, W.A., Revay, E.E., Allan, S.A., Arheart, K.L., Kravchenko, V.D., Xue, R.D., Schlein, Y., Beier, J.C., Müller, G.C., 2013. Attractive toxic sugar baits: control of mosquitoes with the low-risk active ingredient dinotefuran and potential impacts on nontarget organisms in Morocco. Env. Entomol. 42, 1040–1045. Kramer, L.D., Li, J., Shi, P.Y., 2007. West Nile virus. Lancet Neurol. 6, 171–181. Linke, S., Ellerbrok, H., Niedrig, M., Nitsche, A., Pauli, G., 2007. Detection of West Nile virus lineages 1 and 2 by real time PCR. J. Virol. Methods 146, 355–358. Nasci, R., Savage, H.M., White, D.J., Miller, J.R., Cropp, B.C., Godsey, M.S., Kerst, A.J., Bennett, P., Gottfried, K., Lanciotti, R.S., 2001. West Nile virus in overwintering Culex mosquitoes, New York City, 2000. Emerg. Infect. Dis. 7, 742–744. Riabi, S., Gallian, P., Gaaloul, I., Simon, S., Harrath, R., Hassine, M., de Micco, P., Aouni, M., 2010. Prevalence of IgG antibodies against West Nile virus in blood donors during the 2003 outbreak in Tunisia. Trans. Roy. Soc. Trop. Med. Hyg. 104, 507–509. Riabi, S., Gaaloul, I., Mastouri, M., Hassine, M., Aouni, M., 2014. An outbreak of West Nile virus infection in the region of Monastir Tunisia, 2003. Pathog. Global Health 108, 148–157. Rudolf, I., Bakonyi, T., Sebesta, O., Mendel, J., Pesko, J., Betasova, L., Blazejova, H., Venclikova, K., Strakova, P., Nowotny, N., Hubalek, Z., 2014. West Nile virus lineage 2 isolated from Culex modestus mosquitoes in the Czeck Republic, 2013: expansion of the European WNV endemic area to the North? Euro. Surveill. 19, 2–5. Scaramozzino, N., Crance, J.M., Jouan, A., DeBriel, D.A., Stoll, F., Garin, D., 2001. Comparison of flavivirus universal primer pairs and development of a rapid: highly sensitive heminested reverse transcription-PCR assay for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences. J. Clin. Microbiol. 39, 1922–1927. Scherret, J.H., Poidinger, M., Mackenzie, J.S., Broom, A.K., Deubel, V., Lipkin, W.I., Briese, T., Gould, E.A., Hall, R.A., 2001. The relationships between West Nile and Kunjin viruses. Emerg. Infect. Dis. 7, 697–705. Schuffenecker, I., Peyrefitte, C.N., el Harrak, M., Murri, S., Leblond, A., Zeller, H.G., 2005. West Nile virus in Morocco, 2003. Emerg. Infect. Dis. 11, 306–309. Sotelo, E., Fernández-Pinero, J., Vázquez, A., Moreno, A., Aqüero, M., Tenorio, A., Jiménez-Clavero, M.Á., 2011. Phylogenetic relationships of Western Mediterranean West Nile virus strains (1996–2010) using full-length genome sequences: single or multiple introductions? J. Gen. Virol. 92, 2512–2522. Tamba, M., Bonilauri, P., Bellini, R., Calzolari, M., Albieri, A., Sambri, V., Dottori, M., Angelini, P., 2011. Detection of Usutu within a West Nile virus surveillance program in Northern Italy. Vector-Borne Zoonotic Dis. 11, 551–557. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2007. MEGA5. Molecular evolutionary genetics analysis using maximum likelihood evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Triki, H., Murri, S., Le Guenno, B., Bahri, O., Hili, K., Sidhom, M., Dellagi, K., 2001. West Nile viral meningoencephalitis in Tunisia. Med. Trop. 61, 487–490. Turell, M.J., 2012. Members of the Culex pipiens complex as vectors of viruses. J. Am. Mosq. Control Assoc. 28 (Suppl. 4), 123–126. Vázquez, A., Ruiz, S., Herrero, L., Moreno, J., Molero, F., Magallanes, A., Sánchez-Seco, M.P., Figuerola, J., Tenerio, A., 2011. West Nile and Usutu viruses in mosquitoes in Spain, 2008–2009. Am. J. Trop. Med. Hyg. 85, 178–181.
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short communication
West Nile virus in Tunisia, 2014: First isolation from mosquitoes F. Wasfi a , K. Dachraoui a , S. Cherni a , A. Bosworth b , W. Barhoumi a , S. Dowall b , I. Chelbi a , M. Derbali a , Z. Zoghlami a , J.C. Beier c , E. Zhioua a,∗ a b c
Institut Pasteur de Tunis, Laboratory of Vector Ecology, 13 Place Pasteur, 1002 Tunis, Tunisia Public Health England, Porton Down, Salisbury, Wiltshire SP4 0JG, UK Department of Public Health Sciences, University of Miami Miller School of Medicine, Miami, FL 33136, USA
a r t i c l e
i n f o
Article history: Received 11 December 2015 Received in revised form 29 March 2016 Accepted 30 March 2016 Available online 31 March 2016 Keywords: Flaviviruses West Nile virus Culex mosquitoes Emerging North Africa
a b s t r a c t Several outbreaks of human West Nile virus (WNV) infections were reported in Tunisia during the last two decades. Serological studies on humans as well as on equine showed intensive circulation of WNV in Tunisia. However, no virus screening of mosquitoes for WNV has been performed in Tunisia. In the present study, we collected mosquito samples from Central Tunisia to be examined for the presence of flaviviruses. A total of 102 Culex pipiens mosquitoes were collected in September 2014 from Central Tunisia. Mosquitoes were pooled according to the collection site, date and sex with a maximum of 5 specimens per pool and tested for the presence of flaviviruses by conventional reverse transcription heminested PCR and by a specific West Nile virus real time reverse transcription PCR. Of a total of 21 pools tested, 7 were positive for WNV and no other flavivirus could be evidenced in mosquito pools. In addition, WNV was isolated on Vero cells. Phylogenetic analysis showed that recent Tunisian WNV strains belong to lineage 1 WNV and are closely related to the Tunisian strain 1997 (PAH 001). This is the first detection and isolation of WNV from mosquitoes in Tunisia. Some areas of Tunisia are at high risk for human WNV infections. WNV is likely to cause future sporadic and foreseeable outbreaks. Therefore, it is of major epidemiological importance to set up an entomological surveillance as an early alert system. Timely detection of WNV should prompt vector control to prevent future outbreaks. In addition, education of people to protect themselves from mosquito bites is of major epidemiological importance as preventive measure against WNV infection. © 2016 Published by Elsevier B.V.
1. Introduction In the Western Mediterranean basin, West Nile virus (WNV) is transmitted mainly by Culex mosquitoes (Turell, 2012; Vázquez et al., 2011). In natural foci, the circulation of WNV involves birds as amplifying host and bird-feeding mosquitoes (Jourdain et al., 2007) with humans and equines as incidental hosts. WNV is widely distributed in Africa, Middle East, Asia, Southern Europe and the Americas (Hubálek and Halouzka, 1999). A specific WNV lineage 1 variant was also reported in Australia (Scherret et al., 2001). Most of human WNV infections are asymptomatic with less than 1% of infected individuals developing severe neuroinvasive diseases such as meningitis, encephalitis and flaccid paralysis (Kramer et al., 2007).
In Tunisia, several major outbreaks of human WNV infections occurred during 1997 (Triki et al., 2001), 2003 (Hachfi et al., 2010; Riabi et al., 2014), and 2012 (Bouatef et al., 2012). Sporadic cases were reported in 2007, 2010, and 2011 (Bouatef et al., 2012). Several studies showed an intensive circulation of WNV among humans (Riabi et al., 2010; Bahri et al., 2011), equines (Ben Hassine et al., 2014) and birds in Tunisia (Hammouda et al., 2015). A risk map for WNV infection in equines in Tunisia showed that the governorates of Jendouba, Nabeul, Sousse, Monastir, Sfax, Mednine and Djerba are considered as high-risk areas (Bargaoui et al., 2015). To date, no virological data concerning WNV in mosquitoes for Tunisia are available. In the present study, we performed a preliminary virological screening of mosquitoes for WNV in Tunisia. 2. Materials and methods
∗ Corresponding author at: Institut Pasteur de Tunis, Laboratory of Vector Ecology, 13 Place Pasteur BP 74, 1002 Tunis, Tunisia. E-mail addresses: [email protected], [email protected] (E. Zhioua). http://dx.doi.org/10.1016/j.actatropica.2016.03.037 0001-706X/© 2016 Published by Elsevier B.V.
2.1. Study sites and mosquito samplings The study was performed in two villages [(Saddaguia: 35◦ 05 N, 9◦ 25 E and El Felta: 35◦ 16 N, 9◦ 26 E)] belonging to the governorate
F. Wasfi et al. / Acta Tropica 159 (2016) 106–110
107
Fig. 1. Bioclimatic map showing mosquitoes capture sites in Tunisia.
of Sidi Bouzid located in Central Tunisia with an arid bio-climate (Fig. 1). During the last 20 years, environmental changes due to irrigation have occurred in these arid areas located in Cen-
tral Tunisia and lead to the establishment of sand fly vectors of zoonotic visceral leishmaniasis previously limited to the humid areas located in Northern Tunisia (Barhoumi et al., 2015), and to
108
F. Wasfi et al. / Acta Tropica 159 (2016) 106–110
intense circulation of sandfly-borne phleboviruses (Fares et al., 2015). Human-induced habitat change such as the development of irrigation in arid and semi-arid areas of North Africa was shown to have a direct impact on the geographical distribution of C. pipiens (Conley et al., 2014). Within this context, we also investigated the presence of flaviviruses in mosquito populations in these highly environmentally-disturbed areas. Mosquito sampling was performed by using CDC light-traps during September 2014 (September 9, 10, and 23). CDC light-traps were placed inside houses and in animal shelters located in the peri-domestic areas to collect endophilic and exophilic mosquito species. Live mosquitoes were morphologically identified to species level using the identification keys (Brunhes et al., 2000) and were pooled according to the collection site, date, species and sex with a maximum of 5 specimens per pool and directly stored at in −80 ◦ C until use. 2.2. Viral detection, sequencing and phylogenetic analysis Mosquito pools were manually ground in 500 l of Minimum Essentiel Medium (MEM) supplemented with antibiotic solution. The mixture was clarified by centrifugation at 6000g for 2 min. Viral RNA was extracted from 140 l of mosquito homogenates using the QIAmp Viral RNA Mini Kit (Qiagen, Hilden, Germany). During the RNA extraction, no positive control was used to preclude any risk of contamination. Instead, only negative control consisting of water solution was used. RNA extracts were tested by conventional reverse transcription heminested PCR (Scaramozzino et al., 2001) and by a specific West Nile virus real time reverse transcription PCR (Linke et al., 2007). West Nile virus RNA of the Tunisian strain (PaH001) isolated from a deceased patient in 1997 was used as positive control (Triki et al., 2001; Charrel et al., 2003). PCR amplification products were sequenced directly using the Big Dye Terminators v3.1 kit (Applied Biosystems). Sequences of heminested PCR product were firstly aligned together with 23 sequences of selected flaviviruses and with 38 sequences of West Nile virus retrieved from the Genbank database using CLUSTAL W 1.4 implemented in MEGA v.5.22 (Tamura et al., 2007). Phylogenetic analysis was performed based on 213-nt of the NS5 viral polymerase gene. The phylogenetic tree was obtained by using the maximum likelihood analysis method and the tamura-3 model with general time-reversible rates among sites. The tree topology was supported by 500 bootstrap replicates. The Usutu (UsuV) sequence was used as an out-group with West Nile virus sequences. 2.3. Virus isolation Mosquito homogenates of WNV-PCR positive samples were inoculated into Vero cell culture (Vero: WHO cat N◦ 88020401). Only negative control was used to avoid cytotoxicity and contamination risk. Two passages were performed with the Vero cell line. Cells were monitored daily for 7 days for cytopathic effect (CPE). Heminested PCR was performed after only one passage on cells to confirm the presence of detected flavivirus. 3. Results A total of 102 female mosquitoes were collected during 3 trapping nights. All collected mosquitoes from inside houses and animal shelters located in the peri-domestic areas were identified as Culex pipiens. Of a total of 21 pools, 7 were positive by reverse transcription heminested PCR and by the specific West Nile virus real time reverse transcription PCR yielding a minimum infection rate (MIR) of 6.8% (7/102). All positive mosquitoes were collected from animal shelters located in the peri-domestic areas. All PCR products were sequenced and were identified as WNV by Blast analysis. Only RNA
of lineage 1 was detected. Phylogenetic analysis (Fig. 2) showed that the seven sequences of this study formed a monophyletic group supported by an 81% bootstrap value. These sequences were most closely related to the Italian mosquito WNV strain isolated from mosquitoes in 2008, but clearly distinct from the Tunisian WNV strain PHA001 isolated in 1997. Among seven RT-PCR positive pools, only one mosquito homogenate with the lowest ct value inoculated to Vero cells showed CPE. Subsequently, a second RTPCR performed on cell culture supernatant confirmed the presence of WNV viral RNA.
4. Discussion It appears that C. pipiens from North Africa is exo-endophilic species which is a different behaviour from those of Southern Europe where the mosquitoes is mainly exophilic. More studies concerning the behaviour of C. pipiens from North Africa are needed. This report showed the circulation and first detection of WNV lineage 1 in mosquitoes and the first isolation of WNV lineage 1 from mosquitoes in Tunisia. Our results showed that the strain of WNV clusters with the Italian isolate. It is important to point out that our phylogenetic analysis was performed based on a partial highly conserved region of the NS5 polymerase gene (Scaramozzino et al., 2001) allowing only rapid identification of WNV lineages. However, this method has little power to characterize closely related sequences such as the Israeli and North American WNV strains (Sotelo et al., 2011). The clustering of the Tunisian WNV strains with the Italian isolates strongly suggest that WNV is flowing between Tunisia and Europe particularly Italy along migratory bird routes (Jourdain et al., 2007). Our results suggest that C. pipiens is the main vector of WNV in Tunisia. The MIR reported in Czech Republic, Spain, Portugal, and Italy, were 0.012% (Rudolf et al., 2014), 0.1% (Vázquez et al., 2011), 0.28% (Esteves et al., 2004), and 0.02% (Tamba et al., 2011), respectively. In our study, we reported an unexpectedly high MIR but sample size limitation precluded statistical analysis of these data. It is important to point out that this high MIR was obtained by using two different protocols, and therefore, laboratory contamination is excluded. In order to determine a statistically accepted MIR, several thousands of mosquitoes need to be collected and tested for the presence of flaviviruses. Despite the unexpectedly high MIR found in our study, no human cases were reported during 2014 suggesting an intense circulation of WNV among birds. Our results strongly suggest that WNV remains silent and spillover to humans occurs only under favorable ecological conditions. Thus, understanding of the ecological factors to virus circulation and environmental conditions leading WNV outbreaks is of major importance. Persistence of WNV in overwintering C. pipiens is an important mechanism in the maintenance of this arbovirus (Nasci et al., 2001). Thus, WNV is likely to cause future sporadic and foreseeable outbreaks not only in Tunisia but along the migratory flyways of birds between Africa and Eurasia. In addition, outbreaks of WNV infections in humans were reported in Northern Italy, Southern France, Southern Portugal and Central Europe during the transmission season of 2015 (ECDC, 2015). Our results strongly suggest that irrigation in arid areas of North Africa may lead to the emergence of WNV. This hypothesis is corroborated by the intense WNV activity following the development of irrigation networks to flood rice fields located in Northern Morocco (Figuerola et al., 2009; Schuffenecker et al., 2005). Thus, setting up an entomological surveillance as an early alert system around the Mediterranean Basin to prevent future outbreaks is highly needed. New vector control methods, such as attractive toxic sugar bait, show promise for reducing mosquito populations (Khallaayoune et al., 2013), and subsequently should be evaluated in light of our findings.
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Fig. 2. Phylogenetic analysis including 38 different WNV partial NS5 genes and the 7 WNV identified sequences from Tunisian mosquito pools. The Usutu (UsuV) sequence was used as an out-group. Phylogenetic analysis was performed using the maximum likelihood analysis method and the tamura-3 model with general time-reversible rates among sites. The tree topology was supported by 500 bootstrap replicates.
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